Targeted antitoxin delivery platforms as post-exposure therapies for botulism

靶向抗毒素递送平台作为肉毒杆菌中毒的暴露后疗法

• 批准号: 7325538
• 负责人: Brenda A. Wilson
• 金额: $ 40.65万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7325538
• 关键词: Address; Affect; Affinity; Antibodies; Antidotes; Antitoxins; Autonomic nervous system; Binding; Bioterrorism; Blood Circulation; Bontoxilysin; Botulinum Toxin Type A; Botulism; Breathing; C-terminal; Cells; Chimera organism; Chimeric Proteins; Clostridial Neurotoxin; Cytosol; Disease; Facility Construction Funding Category; Food; Future; Goals; Human; Immunization; Ingestion; Intoxication; Length; Light; Location; Mediating; Membrane; Metalloproteases; N-terminal; Nerve; Neuromuscular Junction; Neurons; Neurotoxins; Outcome; Paralysed; Patients; Peptide antibodies; Peptides; Pharmaceutical Preparations; Physiological; Proteins; Reagent; Recombinants; Recovery; Serotyping; Serum; Severity of illness; Specificity; Symptoms; Synaptic Vesicles; System; Therapeutic; Time; Toxin; Vesicle; Work; antibody inhibitor; base; botulinum toxin type B; botulinum toxin type C; botulinum toxin type E; cell type; clinical effect; concept; design; inhibitor/antagonist; neuromuscular function; presynaptic; prevent; receptor; receptor binding; response; scaffold; small molecule; uptake

DESCRIPTION (provided by applicant): One of the greatest challenges of developing post-exposure treatments for botulism caused by clostridial neurotoxins is the brief therapeutic window for conventional antibody-based antitoxins to neutralize the free neurotoxin in the circulation and to block binding of the toxin to cellular receptors. Because of this, the patient is most often asymptomatic until after the toxin has already been internalized and is no longer accessible to antibody neutralization. No current antitoxin is effective after toxin has been internalized and symptoms manifested. Our proposed post-exposure antitoxin will address this critical need. We propose to develop a post-exposure therapeutic anti-botulinum neurotoxin (BoNT) reagent, which builds on the concept of an antibody/inhibitor-toxin chimera that can be targeted for specific delivery into neuronal cells and that upon entry into the cells will deliver and release an inhibitor into the cytosol that will neutralize the catalytic activity of BoNT, such that the cells can more rapidly recover. In this chimeric antitoxin, high affinity scFv antibodies, peptide-based inhibitors, or small molecule-based inhibitors will be attached to the heavy chain of BoNT (BoNT-HC), such that it could specifically inhibit the metalloprotease activity of BoNT-LC after specific receptor binding, translocation (i.e. delivery), and release into the cytosol of neuronal cells. The advantage of using this system is that the neutralizing antitoxin will be directed to the same location where toxin action is occurring - not only the same type of cells, but also the same intracellular compartments where the target substrates are located. Importantly, our proposed antitoxin strategy has the potential to reverse and/or shorten the duration of the clinical effects of intoxication (i.e. paralysis). To achieve the goal of obtaining this new post-exposure antitoxin therapeutic, we propose the following aims: 1. Construction and optimization of recombinant BoNT heavy chain fusion proteins as delivery vehicles. 2. Optimization of the cargo release mechanism for efficient delivery of inhibitor to the cytosol of neuronal cells. 3. Optimization of the substrate-scFv inhibitor as a scaffold for neutralization of BoNT activity. 4. Construction and optimization of alternative BoNT/A-HC adaptors for delivering peptide-based and small-molecule-based inhibitors.

描述（由申请人提供）：开发梭菌神经毒素引起的肉毒中毒暴露后治疗的最大挑战之一是常规抗体抗毒素中和循环中游离神经毒素并阻断毒素与细胞受体结合的短暂治疗窗口。正因为如此，患者通常是无症状的，直到毒素已经被内化，不再能被抗体中和。在毒素被内化并出现症状后，目前没有抗毒素有效。我们提出的暴露后抗毒素将解决这一关键需求。我们建议开发一种暴露后治疗性抗肉毒杆菌神经毒素（BoNT）试剂，其建立在抗体/肉毒杆菌毒素嵌合体的概念上，所述嵌合体可以靶向特异性递送到神经元细胞中，并且在进入细胞后将递送和释放抑制剂到细胞溶质中，所述抑制剂将中和BoNT的催化活性，使得细胞可以更快地恢复。在该嵌合抗毒素中，高亲和力scFv抗体、基于肽的抑制剂或基于小分子的抑制剂将连接到BoNT（BoNT-HC）的重链，使得其可以在特异性受体结合、易位（即递送）和释放到神经元细胞的胞质溶胶中之后特异性抑制BoNT-LC的金属蛋白酶活性。使用该系统的优点是，中和抗毒素将被引导至毒素作用发生的相同位置-不仅是相同类型的细胞，而且是靶底物所在的相同细胞内区室。重要的是，我们提出的抗毒素策略有可能逆转和/或缩短中毒（即麻痹）的临床效应的持续时间。为了实现获得这种新型暴露后抗毒素治疗剂的目标，我们提出了以下目标：1。作为递送载体的重组BoNT重链融合蛋白的构建和优化。2.优化的货物释放机制，有效地传递抑制剂的神经元细胞的胞质溶胶。3.底物-scFv抑制剂作为用于中和BoNT活性的支架的优化。4.构建和优化用于递送基于肽和基于小分子的抑制剂的替代BoNT/A-HC衔接子。